There is currently considerable interest in the process of programmed cell death, known as "apoptosis." This process is considered to be a normal part of an organism's biological defense system. For example, when cells become infected with certain viruses, the resulting overstimulation of the cellular machinery appears to trigger apoptosis; the infected cells die, and thus protect the remaining cells from infection. It is also believed that apoptosis is an important weapon in the body's defense against cancerous growths, and that cancer results at least in part from a loss of the ability of affected cells to trigger their own death.
Researchers often wish to determine whether cells have died because of apoptosis, or because of some other cause. This is generally determined by examining the DNA of the cells. The DNA of cells that have died from apoptosis is typically broken into quite small and uniquely sized fragments, which is generally not the case when cells die from other causes. The DNA fragmentation that is characteristic of apoptosis is caused by enzymes in the cells known as endonucleases, and results in fragments of approximately 300 kilobases and 50 kilobases in length. Often, breaks in the DNA occur in sections between the nucleosome proteins, leading to broken pieces of DNA 180-200 base pairs long (Arends, Morris & Wyllie 1990, Compton 1992, Oberhammer et al. 1993, Walker et al. 1994, Wyllie, Kerr & Curie 1980).
To determine whether apoptosis has occurred, the DNA strand breaks need to be labeled in some manner so that they can be detected. Cell samples are generally first fixed with a crosslinking fixative, so that the small fragments of DNA are not lost from the sample cells in subsequent washing steps. The cells are then treated to make them permeable to further reagents. In two prior art methods, the permeabilized cells are then reacted with deoxynucleotides that have been labeled with either biotin or digoxygenin, using the enzymes DNA polymerase (the "nick translation" enzyme) or terminal deoxynucleotidyl transferase (TdT) to attach the labeled nucleotides to the 3'OH ends of the DNA fragments (Darzynkiewicz et al. 1992, Gorczyca et al. 1992; Gavrieli et al. 1992, Gorczyca, Gong & Darzynkiewicz 1993, Wijksman et al. 1993). The biotin or digoxygenin nucleotides themselves are not readily detectable; however, biotin can be specifically bound by the lectin avidin, and antibodies that can specifically bind digoxygenin are also available. By binding avidin or digoxygenin antibodies to the fluorescent compound fluorescein, which can be done by reacting them with fluorescein-5-isothiocyanate (FITC), and then using these as secondary labels, the DNA strand breaks can be visualized by observing the fluorescence of the labeled DNA strand ends. A single step method utilizing deoxynucleotides directly labeled with fluorochromes, which is simpler but less sensitive than the above-described indirect methodology, has also been recently described (Gold et al. 1993, Li, et al. 1995).
Another area of active research is the study of DNA replication and repair. One method for detecting the cellular replication and/or repair of DNA is known as Strand Breaks Induced by Photolysis, or SBIP. In this method, the living cells are first supplied with halogenated DNA precursors such as BrdUrd or IdUrd, which the cells will incorporate into the DNA during replication or repair. The cells are then exposed to ultraviolet light, which causes the DNA to break where the halogenated precursors have been incorporated. The resulting DNA strand breaks can then be detected by fluorochrome labeled antibodies, as described above (Li, Traganos & Darzynkiewicz, 1994, Li et al. 1994c, 1995).
A third important use for DNA strand labeling is perhaps the oldest and most widely practiced; end-labeling of DNA strands during the purification and characterization of DNA, so that the DNA can be detected. As well appreciated by those in the field, DNA research samples are generally too small even to be seen with the naked eye. In order to detect them, they are often "end labeled" at a very early stage of purification. For example, it is common to end label DNA with nucleotides containing radioactive phosphorous (P.sup.32); the presence of the DNA can then be detected at various stages of analysis using a geiger counter, by scintillation counting or by autoradiography. However, such methods are currently being phased out due to the biological hazards involved in working with radioactive compounds, and because of the ecological problems caused by the need to dispose of such radioactive materials. These methods are being replaced largely by methods that provide for labeling with fluorescent compounds. However, methods in use prior to the present invention are often complex, and are generally somewhat expensive.